The long term goal of the proposed research is to define the precise mechanistic steps employed to ensure the structural maturation of multi-pass membrane proteins as they are synthesized by the ribosome and are integrated into the lipid bilayer. Relevance to disease is especially apparent in cystic fibrosis, which is caused by mutations in the polytopic protein CFTR, leading to its misfolding and, therefore, premature degradation. Intelligent design of disease treatment ultimately relies upon our ability to identify the biosynthetic event gone awry with disease-causing mutant proteins. Using a model polytopic protein, aquaporin 4 (AQP4), the current proposal determines the manner in which transmembrane segments move from the endoplasmic reticulum Sec61 translocon pore into the lipid bilayer, a fundamental phase during the biogenesis of polytopic proteins. Additionally, the functional organization of the translocon during distinct stages of AQP4 biogenesis will be defined using biochemical measures, namely by determining ribosome-translocon complex stability and composition at defined stages. These studies will, thereby, provide valuable information as to how the translocon proceeds step-wise to assemble functional polytopic proteins. PUBLIC HEALTH RELEVANCE One step in the creation of disease treatment is the investigation of how relevant protein mutations lead to the protein's malfunction. Cystic fibrosis is caused by mutations in a membrane bound protein that prevents its structural maturation into a functional protein. This research elucidates mechanisms by which membrane proteins acquire full structural maturation, thereby, providing a framework to examine disease-related mutations in membrane bound proteins.